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Identification of transglutaminase substrates and uses therefor

a transglutaminase and substrate technology, applied in the direction of transferases, peptide sources, plasma/lacto globulins, etc., can solve the problems of difficult finding common substrate motifs, for example transglutaminase substrate motifs, and limited identification of synthetic peptides as enzyme substrates, so as to achieve optimal enzyme specificity

Active Publication Date: 2022-03-08
ROCHE SEQUENCING SOLUTIONS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patent describes a way to quickly and easily create a library of peptides that can be used as substrates for an enzyme called transglutaminase. This library is made by synthesizing millions of small peptides on a special array and then testing them to see which ones are the best substrates for the enzyme. This approach allows for a comprehensive and reproducible screening of transglutaminase substrates, and it also allows for the rapid creation of new arrays to search for even more peptide substrates.

Problems solved by technology

One challenge is that the identification of synthetic peptides as enzyme substrates is limited by the potentially large number of molecules that could be tested.
However, the wide diversity of sequences makes finding common substrate motifs, for example transglutaminase substrate motifs, difficult.

Method used

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  • Identification of transglutaminase substrates and uses therefor
  • Identification of transglutaminase substrates and uses therefor
  • Identification of transglutaminase substrates and uses therefor

Examples

Experimental program
Comparison scheme
Effect test

example 1

elopment

[0232]To test MTG (Zedira GmbH) specificity for Gln-substrate, N-(Biotinyl)cadaverine (Zedira GmbH) was used as a substitute for a Lys-substrate to biotinylate Gln-peptides on a peptide array synthesized using maskless light-directed peptide array synthesis. Similarly, to test MTG specificity for Lys-substrate, Z-Gln-Gly-CAD-Biotin (Zedira GmbH) was used as a substitute for a Gln-substrate to biotinylate Lys-peptides. Z-Gln-Gly-CAD-Biotin is a glutamine donor substrate for transglutaminases having the following formula:

[0233]

[0234]After treatment with MTG in the presence of one of the biotinylated substrates, arrays were washed, stained with Cy5-streptavidin to label biotin moieties, and scanned at 635 nm to measure signal intensity at the peptide areas. Signal intensity corresponding to the efficiency of MTG reaction was used to determine specificity of different peptide sequences.

example 2

ditions

[0235]In general, there were two issues to consider in optimizing array performance of the enzymatic assay originally developed under solution conditions. A first issue was a challenge of low signal generation that could be caused by the inability of the enzyme to recognize peptides bound to the surface, low peptide concentration, insufficient quality of peptide synthesis, or by surface effect on enzyme stability and reactivity. A second issue was a challenge of high background generation that may be a result of non-specific binding of the enzyme and / or a substrate to an array surface or non-specific labeling driven by side reactions on the array.

[0236]To find conditions for MTG assay on the array, the effects of various parameters of the reaction on signal and background generation were analyzed. These factors included MTG and substrate concentrations, reaction buffer composition, and incubation time and temperature. In order to minimize background, a non-protein blocking so...

example 3

ficity for Gln-Peptides

[0238]A 5-mer peptide array was incubated in the presence of MTG and biotinylated amine donor N-(Biotinyl)cadaverine substrate under conditions described in Example 2. Signal distribution and correlation between two replicates was plotted (FIG. 1).

[0239]With reference to FIG. 1, correlation in signal intensity between replicates showed high reproducibility of the data and allowed identification of peptides with the highest labeling efficiency. Sequences and corresponding signal intensity of 25 peptides with the highest labeling efficiency in an array MTG assay with biotinylated amine-donor substrate discovered in this study are shown in Table 2. In concordance with MTG specificity, all peptides contained a Gln (Q) residue. Gln occupied exclusively the fifth position in all selected sequences. Because the 5-mer peptides synthesized on the array were flanked by G:S linkers for which a 3:1 mixture of Gly and Ser amino acid precursors was used, Gln may be followed...

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Abstract

According to one aspect, the present disclosure provides a method of identifying a substrate of a transglutaminase using a peptide array comprising a plurality of peptides. The method includes the steps of contacting the peptides in the peptide array with the transglutaminase, allowing the transglutaminase to bind to the peptides, and identifying the substrate of the transglutaminase.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This patent application is a continuation of International Patent Application No. PCT / EP2015 / 079689 filed Dec. 15, 2015, which claims priority to and the benefit of U.S. Provisional Application No. 62 / 094,495, filed Dec. 19, 2014. Each of the above patent applications is incorporated herein by reference as if set forth in its entirety.BACKGROUND OF THE INVENTION[0002]The disclosure relates, in general, to peptide arrays comprising transglutaminase substrates, and methods of identifying transglutaminase substrates using peptide arrays. The disclosure also relates to transglutaminase substrate peptides and methods of their use to cross-link peptides and proteins.[0003]Elucidating the details of enzyme activity and specificity is important for understanding the physiological function of enzymes and for biotechnological applications of the reactions catalyzed by enzymes. For example, transglutaminases belong to a large family of related enzym...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K16/28C07K16/30A61K38/08C12P21/02C12N9/10C07K14/47C07K1/107C07K1/13C07K14/00
CPCC12P21/02C07K1/1075C07K1/13C07K14/00C07K14/4717C12N9/1044C07K2319/21C07K7/06
Inventor ALBERT, THOMASBERGMANN, FRANKLYAMICHEV, VICTORPATEL, JIGARSCHRAEML, MICHAELSTEFFEN, WOJTEK
Owner ROCHE SEQUENCING SOLUTIONS INC
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